Comparison of different techniques for semiquantitative detection of HBV DNA by PCR

Conclusions

Our results indicate that the choice of the probe for ELOSA is of major concern. In our panel we had seven sera which contained about 100 molecules/ml and further five sera which contained less than 1000 molecules/ml. Most of them were detected by the long probe but not by the short probe. When PCR for the S-or PreS-gene was included it was possible to detec all 24 HBV-positive sera (not shown) by ELOSA. The reliable lower quantification limit for the long probe is 250 molecules/ml and for the short probe 2500 molecules/ml. Surprisingly, chemiluminescence did not produce better qualitative or quantitative results. The data suggest that the usage of several replicates allows relative quantification in most cases. One possible drawback we see is the hybridization efficiency. Six of our positive samples showed great differences between the number of target molecules suggested by agarose gel electrophoreses or by hybridization (Southern blot or ELOSA). All of them contained more than 10⁶ molecules/ml. For these cases and for the samples where the short probe and the long probe gave discordant result (2 cases) we think that competitive PCR will be the method of choice, but in most cases ELOSA with the long probe gives reliable results and is highly sensitive.